Hypermethylation of serotonin transporter gene in bipolar disorder detected by epigenome analysis of discordant monozygotic twins.

Bipolar disorder (BD) is a severe mental disorder characterized by recurrent episodes of mania and depression. Serotonin transporter (HTT) is a target of antidepressants and is one of the strongest candidate molecules of mood disorder, however, genetic study showed equivocal results. Here, we performed promoter-wide DNA methylation analysis of lymphoblastoid cell lines (LCLs) derived from two pairs of monozygotic twins discordant for BD. To rule out the possible discordance of copy number variation (CNV) between twins, we performed CNV analysis and found the copy number profiles were nearly identical between the twin pairs except for immunoglobulin-related regions. Among the three genes we obtained as candidate regions showing distinct difference of DNA methylation between one of the two pairs, hypermethylation of SLC6A4, encoding HTT, in the bipolar twin was only confirmed by bisulfite sequencing. Then, promoter hypermethylation of SLC6A4 in LCLs of BD patients was confirmed in a case-control analysis. DNA methylation of SLC6A4 was significantly correlated with its mRNA expression level in individuals with the S/S genotype of HTTLPR, and mRNA expression level was lower in BD patients carrying the S/S genotype. Finally, DNA methylation of the same site was also higher in the postmortem brains of BD patients. This is the first study to report the role of epigenetic modification of SLC6A4 in BD using an unbiased approach, which provides an insight for its pathophysiology.

Characterization of a novel member of the FGF family, XFGF-20, in Xenopus laevis.

The cDNA for a novel member of the FGF family (XFGF-20) was isolated from a Xenopus cDNA library prepared at the tailbud stage using as a probe the product of degenerate PCR performed with primers based on mammalian FGF-9s. This cDNA was 1860 bp long, and contained a single open reading frame that encoded 208 amino acid residues. The deduced amino acid sequence contained a motif characteristic of the FGF family and it was similar (73.1% overall homology) to XFGF-9 but differed from XFGF-9 in its amino-terminal region (33.3% homology). XFGF-20 mRNA was expressed only zygotically in embryos at and after the blastula stage, but it was also specifically expressed in the stomach and testis of adults. By contrast, XFGF-9 mRNA was expressed maternally in eggs and in many adult tissues. When XFGF-20 mRNA was overexpressed in early embryos, gastrulation was abnormal and development of anterior structures was suppressed. In such embryos, the expression of the Xbra transcript was suppressed during gastrulation while the expression of the transcripts of cerberus, Siamois, dkk-1, chordin, and Xotx-2 genes was normal. These results suggest that correct expression of XFGF-20 during gastrulation is required for the formation of normal head structures in Xenopus laevis during embryogenesis and that expression of the Xbra gene mediates this phenomenon.

Human genes for KNSL4 and MAZ are located close to one another on chromosome 16p11.2.

KNSL4 (Kid; kinesin-like DNA-binding protein) is a member of the kinesin family that is involved in spindle formation and the movements of chromosomes during mitosis and meiosis. Myc-associated zinc finger protein (MAZ) participates in both the initiation and the termination of transcription of target genes. We isolated genomic DNA clones that encoded KNSL4 and MAZ from a human cosmid library. Sequence analysis revealed that the two genes were very close to one another. The distance between the two genes was only 1. 2 kb, and this intervening 1.2-kb region was extremely GC-rich. The gene for KNSL4 spanned 16 kb and consisted of 14 exons and 13 introns, while the gene for MAZ spanned 6 kb and consisted of 5 exons and 4 introns. The two genes were mapped to chromosome 16p11.2 by fluorescence in situ hybridization.

Isolation and expression study of a maternally expressed novel Xenopus gene Xem1 encoding a putative evolutionarily conserved membrane protein.

A novel Xenopus maternally expressed gene, Xem1, was isolated by differential display PCR and 5'-RACE. Xem1 coded for a putative transmembrane protein of 172 amino acids. Sequence analysis, including the clustering and reconstruction of ESTs (Expressed Sequence Tags), revealed that homologs of Xem1 are widely distributed in eukaryotic phyla, suggesting that Xem1 is a member of evolutionarily conserved proteins. Expression of Xem1 mRNA occurred from the previtellogenic stage and its level increased during oogenesis, maintained throughout oocyte maturation to blastula stage and then decreased in post gastrula stages. In cleavage stage, Xem1 RNA was distributed uniformly, and in adult, occurred predominantly in ovary and testis. We assume that Xenopus Xem1 may have its function in gametogenesis and in early phase of embryogenesis, whose function may be related to transport mechanism of small molecular weight substances like metal ions, from analogy to the function of its homologs in other organisms.

Differential display analysis of gene expression in developing embryos of Xenopus laevis.

Differential display (DD), an arbitrarily primed RT-PCR fingerprinting technique, is a novel approach for the search of differentially expressed transcripts. Using our improved DD protocol, reproducible cDNA fingerprints were successfully obtained from RNAs of Xenopus laevis embryos at six representative stages. Parallel comparison among the fingerprints revealed a number of bands with differential expression patterns. Analysis with clones of three randomly chosen bands confirmed that their expression patterns were faithfully reflected on fingerprints, thereby proving the reliability and validity of the approach. Nucleotide sequencing of these clones revealed that one is identical with a known transcript (cardiac actin), the second is a novel developmentally regulated gene showing no significant homology with those reported previously, and the last is a close but unique relative of XK endo B gene showing somewhat different spatial expression pattern. These results indicated that the DD analysis provides a rapid and reliable way for the identification of novel differentially expressed genes as well as a unique 'scope' for the survey of the changes in overall gene expression profiles occurring in the early embryonic development of Xenopus as well as of other organisms.

Fluorescent differential display: arbitrarily primed RT-PCR fingerprinting on an automated DNA sequencer.

We established robust, reliable protocols for 'Differential Display (DD),' an RNA fingerprinting method originally developed by Liang and Pardee [(1992) Science 257, 967-971] using RT-PCR with arbitrary primers. Our protocols are optimized so that reliable DD analysis can be performed on a fluorescent DNA sequencer to ensure high throughput as well as improved operational safety, compared with the original one using radioactive compounds. Such 'Fluorescent Differential Display (FDD)' techniques will accelerate the identification of differentially expressed as well as polymorphic transcripts to address various biological questions.